Diabetogene rad: a type II diabetes specific gene

ABSTRACT

Purified DNA including a sequence encoding Diabetogene rad.

This application is a continuation-in-part of U.S. Ser. No. 07/901,710,filed on Jun. 19, 1992, now abandoned.

BACKGROUND OF THE INVENTION

The invention relates to the genetic basis of diabetics.

Diabetes mellitus is among the most common of all metabolic disorders,affecting up to 11% of the population by age 70. Type I (insulindependent diabetes mellitus or IDDM) diabetes represents about 5 to 10%of this group and is the result of a progressive autoimmune destructionof the pancreatic β-cells with subsequent insulin deficiency. Type II(non-insulin dependent diabetes mellitus or NIDDM) diabetes represents90-95% of the affected population but is much less well understood fromthe point of view of primary pathogenesis. Type II diabetic patientsexhibit elements of both insulin resistance and relative insulindeficiency.

Alterations in glucose homeostasis are the sine qua non of diabetesmellitus and occur in both the Type I and Type II forms of the disease.In the mildest forms of diabetes this alteration is detected only afterchallenge with a carbohydrate load, while in moderate to severe forms ofdisease hyperglycemia is present in both the fasting and postprandialstates. The most important tissue involved in disposal of a glucose loadfollowing oral ingestion, i.e., in the absorptive state, is skeletalmuscle. (Klip 1990 Diabetes Care 13:228-243; Caro et al. 1989 Diab.Metab. Rev. 5:665-689; Bogardus 1989 Diab. Metab. Rev. 5:527-528;Beck-Nielsen 1989 Diab. Metab. Rev. 5:487-493) Skeletal muscle comprises40% of the body mass, but has been estimated to account for between 80and 95% of glucose disposal at high insulin concentration or followingan oral glucose load. (Beck-Nielsen 1989; Baron et al. 1988 Am. J.Physiol. 255:E769-74) In insulin-treated animals, about 25% of anintravenous glucose load enters muscle within 1 minute. (Daniel et al.1975 J. Physiol. (Lond) 247:273-288)

Skeletal muscle takes up glucose by facilitated diffusion in both aninsulin-independent and insulin-dependent manner and has been shown toexpress relatively high levels of GLUT4 (the "insulin responsive"glucose transporter) and low levels of GLUT1 and GLUT3 (the transportersbelieved to be involved in basal glucose transport). (Mueckler 1990Diabetes 39:6-11; Bell et al. 1990 Diabetes Care 13:198-208) Once insidethe muscle, glucose is rapidly phosphorylated by hexokinase to formglucose 6-phosphate. Although the rate-limiting step for glucose uptakeis at the level of transport, there is increasing evidence that themajor control of carbohydrate metabolism is exerted after theglucose-6-phosphate step. (Mandarino 1989 Diab. Metab. Rev. 5:474-486;Felbert et al. 1977 Diabetes 26:693-699) Depending on the hormonalmilieu and metabolic state, the glucose 6-phosphate can enter eitheranabolic or catabolic pathways. The major anabolic pathway involvesconversion of the glucose to glycogen. The rate-limiting enzyme of thisreaction is glycogen synthase. The activity of glycogen synthase isregulated primarily by phosphorylation and dephosphorylation and thepresence of the allosteric regulator glucose-6-phosphate, although thelevel of expression of the enzyme must also play a role. In catabolicstates, glucose is metabolized through the glycolytic pathway topyruvate which in turn is either converted to lactate (under anaerobicconditions) or is oxidized by CO₂ and acetyl-CoA. The latter reaction iscatalyzed by the multienzyme complex pyruvate dehydrogenase (PDH). PDHactivity is also regulated by the level of the enzyme, phosphorylationand dephosphorylation, and a number of allosteric modifiers. Most of theenzymes and proteins involved in glucose metabolism have been identifiedand purified, and over the past several years, several of these havebeen cloned at a molecular level.

The dominant hormone regulating glucose metabolism in muscle is insulin.Insulin exerts its actions through insulin, and to a lesser extentIGF-1, receptors, both of which are expressed in skeletal muscle.(Beguinot et al. 1989 Endocrinology 125:1599-1605; Sinha et al. 1987 J.Clin. Invest. 79:1330-1337; Obermaier-Kusser et al. 1989 J. Biol. Chem.264:9497-9504; Arner et al. 1987 Diabetologia 30:437-440) Like insulinand IGF-1 receptors on other tissues, these receptors are proteintyrosine kinases which are stimulated upon insulin and IGF-1 binding.(White et al. J. Clin. Invest. 82:1151-1156) This initial insulin signalthen acts through a cascade of events involving phosphorylation anddephosphorylation, as well as possible mediator generation to promoteglucose uptake, stimulate metabolism and conversion of glucose toglycogen by activating glycogen synthase, and regulate a variety ofintracellular enzymes involved in carbohydrate. (Yki-Jarvinen et al.1987 J. Clin. Invest. 80:95-100; Mandarino et al. 1987 J. Clin. Invest.80:655-663) In addition, insulin also acts at the level of muscle tomodify lipid and protein metabolism through effects on membranetransport, enzyme activity and gene expression. (Kimball et al. 1988Diab. Metab. Rev. 4:773-787; Alexander et al. 1988 Proc. Natl. Acad.Sci. USA 85:5092-5096)

In both Type I and Type II diabetes there are major alterations in theability of peripheral tissues to take up and metabolize glucose.(DeFronzo 1988 Diabetes 37:667-687; Olefsky et al. 1988 Am. J. Med.85:86-105; Reaven 1988 Diabetes 37:1595-1607; Nankervis et al. 1984Diabetologia 27:497-503; Yki-Jarvinen et al. 1986 N. Engl. J. Med.315:224-230; Hother-Nielsen et al. 1987 Diabetologia 30:834-840) Thesealterations affect liver, fat and muscle, as well as other tissues. InType I diabetes, the alterations in glucose metabolism are largelysecondary to insulin deficiency which has both acute and chronic effectswith regard to regulation of glucose uptake and intracellulardisposition metabolism. (Nankervis 1984; Yki-Jarvinen 1986;Hother-Nielsen 1987) The exact basis for impaired metabolism in muscleof Type II diabetics is less clear, but probably involves a combinationof factors including a significant level of insulin resistance (due toacquired or genetic factors), as well as some component of relativeinsulin deficiency.

In obesity and diabetes, there are a variety of alterations in themuscle glucose homeostasis. In obese individuals without diabetes themajor alteration is in oxidative glucose metabolism. (Beck-Nielsen 1989;DeFronzo 1988; Olefsky 1988) There are reduced insulin-stimulatednonoxidative glucose metabolism, a reduction in both basal andinsulin-stimulated glucose oxidation and a higher rate of lipidoxidation than in lean controls. (Felber et al. 1987 Diabetologia26:1341-1350) Glycogen synthase activity is decreased in obeseindividuals and may contribute to the reduced nonoxidative glucosedisposal. (Bogardus et al. 1984 J. Clin. Invest. 73:1185-1190; Freymondet al. 1988 J. Clin. Invest. 82:1503-1509) In obese diabetics bothoxidative and nonoxidative pathways are altered, and the latter may playa more quantitatively important role. (Beck-Nielsen 1989) In bothobesity and Type II diabetes there is also decreased insulin stimulatedreceptor phosphorylation (Caro 1987; Obermaier-Kusser 1989; Arner 1987)decreased insulin stimulated glucose transport (Caro 1987; Felber 1987)decreased insulin-stimulated pyruvate dehydrogenase activity (Mandarino1989), and defective insulin-stimulated glycogen synthase. (Mandarino1989; Freymond 1988; Thorburn et al. 1990 J. Clin. Invest. 85:522-529)Untreated Type I diabetic humans and rodents show many of the samechanges. (Nankervis 1984; Yki-Jarvinen 1986; Hother-Nielsen 1987;Wallberg 1989 Med. Sci. Sports. Exerc. 21:356-361) In the latter group,these tend to reverse with proper therapy, although in most studies somereduction in insulin-stimulated glucose oxidation and insulin-stimulatedPDH activity persist despite therapy. Factors mediating thesealterations in glucose homeostasis in diabetes and obesity are multipleand include the altered hormonal milieu, altered substrate levels, andpossibly even circulating insulin antagonists. (DeFronzo 1988; Sugden etal. 1990 J. Endocrinol. 127:187-190; Leighton et al. 1990 TrendsBiochem. Sci. 15:295-299) A summary of the defects in glucose metabolismin muscle in Types I and II diabetes and obesity is given in reference4.

Although controversy exists as to the primary defect in Type II diabetesseveral studies suggest that the earliest detectable abnormality may bein glucose disposal by muscle. (Bogardus 1989; DeFronzo 1988; Reaven1988; Lilloja et al. 1988 Acta Med. Scand. Suppl. 723:103-119; Ho et al.1990 Diabetic Med. 7:31-34; Eriksson et al. 1989 N. Engl. J. Med.321:337-343; Bogardus et al. 1989 Diabetes 38:1423-1432; Lilloja et al.1987 Diabetes 36:1329-1335; Knowler et al. 1990 Diab. Metab. Rev.6:1-27; Warram et al. 1990 Ann. Int. Med. 113:909-915; Martin et al.Submitted for publication) In a study initiated by Dr. J. StuartSoeldner over 20 years ago, over 200 offspring of two Type II diabeticparents were identified and evaluated for abnormalities in glucosetolerance, glucose disposal and insulin secretion (Warram 1990; Martin)155 of the offspring were normoglycemic at the onset of study. Theindividuals were subsequently followed for an average of 14 years duringwhich time 25 developed Type II diabetes. Analysis of data gathered atentry to the study provided a unique insight as to the earliest defectsdetectable in prediabetic individuals. This study revealed that therewere no differences in either first or second phase insulin secretorycapacity in these offspring which predicted the development of diabetes.However, overall glucose disposal rate was reduced. Based on the Bergmanmodel of glucose disposal (Bergman 1989 Diabetes 38:1512-1527), thisdecrease in glucose disposal rate was due to reduced insulin sensitivity(S₁) and insulin independent glucose disposal (S_(G)). Low values of S₁and/or S_(G) were highly predictive of the subsequent development ofdiabetes. (Warram 1990; Martin) If one compares the normoglycemicoffspring in this study with the lowest quintile of insulin sensitivityto those offspring in the highest quintile of insulin sensitivity, therewas a 62-fold increase in relative risk of developing Type II diabetesduring follow-up (see preprint of Martin in Appendix). Likewise lowglucose sensitivity (which reflects both insulin-independent and basalinsulin-dependent glucose disposal) increased the relative risk ofdiabetes by 22-fold.

Similar findings and conclusions have been derived from studies of thePima Indian population which has a very high incidence of Type IIdiabetes. (Lilloja 1988; Ho 1990; Erikkson 1989; Bogardus 1989; Lilloja1987; Knowler 1990) In addition, in this population, evidence has beenpresented that insulin sensitivity is inherited in family clusters andwith a pattern of distribution suggestive of an autosomal dominanttrait. (Bogardus 1989; Lilloja 1987) Alterations in glucose metabolismand glycogen synthase activity can also be detected in biopsies fromprediabetic Pima Indians prior to onset of clinical diabetes. (Knowler1990; Warram 1990; Martin; Bergman 1989; Foley 1988 Diabet. Metabl. Rev.4:487-505) Consistent with the hypothesis that Type II diabetes developsin the presence of an underlying defect in insulin sensitivity are themany observations which indicate that aggressive insulin therapy in theType II diabetic may normalize blood glucose and glycohemoglobin, butusually fails to reverse completely the insulin resistance which isobserved in this disease. (DeFronzo 1988; Olefsky 1988) Geneticvariability in insulin sensitivity may also exist in the non-diabeticCaucasian populations (Hollenbeck et al. 1987 J. Clin. Endocrinol.Metab. 64:1169-1173), suggesting that genes which control insulinsensitivity may exist at some frequency in control non-diabetic, as wellas diabetic, population.

Although there is considerable evidence for alterations in themetabolism and the activity of a variety of enzymes and transporters inmuscle in diabetes mellitus, including changes in insulin receptors,glucose transporters, glycogen synthase and pyruvate dehydrogenase,there is less information concerning specific alterations in theexpression of the genes for these proteins or other proteins which mightaccount for the altered insulin action. Alterations in the level of mRNAfor the insulin-sensitive glucose transporter GLUT4, but not GLUT1, havebeen observed in skeletal muscle of streptozotocin diabetic rats and arerestored toward normal by insulin or vanadate treatment. (Bourey 1990 J.Clin. Invest. 86:542-547; Strout et al. 1990 J. Endocrinology126:2728-2732; Sivitz 1989 Nature 340:72-74). Insulin and glucose havealso been shown to regulate glucose transporter mRNA expression in thecultured skeletal muscle cell line L6. (Walker et al. 1989 J. Biol.Chem. 264:6587-6595) In both rodent and human models of obesity and TypeII diabetes, the data on glucose transporter mRNA expression are morecontroversial with some studies showing altered levels of GLUT4expression in adipose tissue, but not muscle, and others showingalterations in both tissues. (Caro 1989; Pedersen 1990 Diabetes39:865-870) Diabetes has also been shown to affect the level ofexpression of insulin-like growth factors I and II in muscle (Leaman1990 Endocrinology 126:2850-2857), creatine kinase (Popovich 1989 Amer.J. Physiol 257:E573-E577), glutamine synthetase (Feng 1990 Am. J.Physiol. 258:E762-E766), and the α-subunit of guaninnucleotideregulatory protein, G_(S). (Griffiths et al. 1990 Eur. J. Biochem.193:357-364) By contrast, mRNA for the important bifunctional enzyme6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is altered in liverof STZ diabetic rats, but is not altered in muscle. (Colosia 1988 J.Biol. Chem. 263:18669-18677) Some investigators have also shown a changein expression of one of the alternatively spliced forms of the insulinreceptor in muscle of human with Type II diabetes, although thisapparently occurs without a significant change in level of totalreceptor mRNA. (Mosthaf et al. 1991 Proc. Natl. Acad. Sci. USA88:4728-4730) Levels of total mRNA and those of most of the majorstructural proteins like actin do not appear to be altered in diabetes.(Pedersen 1990) Little or no data yet exist for effects of diabetes onthe mRNA levels in muscle for glycogen synthase, PDH, hexokinase, theinsulin receptor substrate (IRS-1) etc., although gene probes now existfor a number of these important metabolic enzymes. (Persons et al. 1989Mol. Carcinog. 2:88-94; Dugail 1988 Biochem. J. 254:483-487; Arora etal. 1990 J. Biol. Chem. 65:6481-6488; Minchenko et al. 1984 Endocrinol.Exp. 18:3-18; Sun et al. 1991 Nature 352:73-77)

SUMMARY OF THE INVENTION

In general, the invention features purified DNA including a sequenceencoding the Diabetogene rad. A diabetogene is a gene whose expression,at the mRNA level, is altered (i.e., is different from what is seen in anormal individual) in an individual with diabetes and/or obesity. Radencodes a new member of the ras/GTPase related gene family. Rad shares45-55% homology at the nucleotide level and 33% homology at the aminoacid level with other members of this family in the GTPase domain, buthas additional 5' and 3' coding sequences compared to the low molecularweight GTP-binding proteins. Rad was formerly referred to as DiabetogeneC9D6. The invention also includes: a vector including a DNA sequenceencoding Diabetogene rad; a cell, e.g., a cultured cell, e.g., a musclecell, containing purified Diabetogene rad DNA; a cell capable of givingrise to a transgenic animal; a cell capable of expressing a polypeptideencoded by Diabetogene rad; an essentially homogeneous population ofcells, each of which includes the purified Diabetogene rad DNA.

In another aspect, the invention features a substantially purepreparation of a polypeptide encoded by rad. The invention also includesa purified preparation of an antibody, e.g., a monoclonal antibody,directed against rad.

In another aspect, the invention features a therapeutic compositioncomprising a diabetogene product, e.g., the rad protein, and apharmaceutically acceptable carrier.

In another aspect, the invention features a method for manufacture ofthe rad protein comprising culturing a cell transformed with rad DNA ina medium to express the protein.

In another aspect, the invention features a method for treating ananimal (e.g., a human afflicted with Type I or Type II diabetes, or anexperimental animal, e.g., an animal model for a disease or disorder,e.g., a NOD mouse, an ob/ob mouse, a db/db mouse, a Zucker fatty rat, ora streptozotocin induced rat) having a disease characterized by adeficiency or an excess in a diabetogene product including,administering a therapeutically-effective amount of the diabetogeneproduct (in the case of a deficiency) or an inhibitor of production orfunction of the diabetogene product (in the case of an excess) to theanimal.

In another aspect, the invention features a method of evaluating theeffect of a treatment including administering the treatment andevaluating the effect of the treatment on the expression of adiabetogene. In preferred embodiments, the treatment is administered to:an animal, e.g., a human afflicted with Type I or Type II diabetes; anexperimental animal, e.g., an animal model for a disease or disorder,e.g., a NOD mouse, an ob/ob mouse, a db/db mouse, a Zucker fatty rat, ora streptozotocin induced rat, or a transgenic animal carrying adiabetogene, e.g., a Diabetogene rad transgene; or a cell, e.g. acultured cell, e.g., a cultured muscle cell.

In preferred embodiments: the expression of the diabetogene is increasedin Type II diabetes (and preferably not increased in Type I diabetes);the diabetogene is the gene for muscle glycogen phosphorylase, the geneencoded by clone F208 (see below), or the gene for elongation factor 1α;the expression of the diabetogene is not elevated in Type I diabetes;the diabetogene is Diabetogene rad; the expression of the diabetogene isdecreased in Type II diabetes; the expression of the diabetogene isincreased in normal individuals; the expression of the diabetogene isdecreased in normal individuals; the expression of the diabetogene isincreased in Type I diabetes.

In another aspect, the invention features a method for determining if asubject is at risk for a glucose related disorder, e.g., diabetes orobesity, including examining the subject for the expression of adiabetogene, non-wild type expression being indicative of risk.

In preferred embodiments: the expression of the diabetogene is increasedin Type II diabetes (and preferably not increased in Type I diabetes);the diabetogene is the gene for muscle glycogen phosphorylase, the geneencoded by clone F2D6, or the gene for elongation factor 1α; theexpression of the diabetogene is not elevated in Type I diabetes; thediabetogene is Diabetogene rad; the expression of the diabetogene isdecreased in Type II diabetes; the expression of the diabetogene isincreased in normal individuals; the expression of the diabetogene isdecreased in normal individuals; the expression of the diabetogene isincreased in Type I diabetes.

In another aspect, the invention features a method for determining if asubject is at risk for a glucose related disorder, e.g., diabetes orobesity, including providing a nucleic acid sample from the individualand determining if the structure of a diabetogene differs from wildtype, departure from wild type indicating risk. In preferredembodiments: the determination includes determining if the diabetogenehas a gross chromosomal rearrangement, e.g. by blot analysis; thedetermination includes sequencing the diabetogene; the subject is ahuman; the subject is a NOD mouse, an ob/ob mouse, a db/db mouse, aZucker fatty rat, or a streptozotocin induced rat.

In preferred embodiments: the expression of the diabetogene is increasedin Type II diabetes (and preferably not increased in Type I diabetes);the diabetogene is the gene for muscle glycogen phosphorylase, the geneencoded by clone F2D6, or the gene for elongation factor 1α; theexpression of the diabetogene is not elevated in Type I diabetes; thediabetogene is Diabetogene rad; the expression of the diabetogene isdecreased in Type II diabetes; the expression of the diabetogene isincreased in normal individuals; the expression of the diabetogene isdecreased in normal individuals; the expression of the diabetogene isincreased in Type I diabetes.

In another aspect, the invention features a method of evaluating ananimal model for a disorder or disease state, e.g., a glucose relateddisorder, e.g., diabetes or obesity, including determining if adiabetogene in the animal model is expressed at a predetermined level.In preferred embodiments, the predetermined level is lower than thelevel in wild type or normal animal; the predetermined level is higherthan the level in a wild type or normal animal.

In another aspect, the invention features a transgenic rodent, e.g. amouse, hamster, or rat, having a transgene which includes a diabetogene,e.g., Diabetogene rad. In preferred embodiments, the transgenicdiabetogene is a mutant form, e.g., a deletion mutant, of the gene.

In another aspect, the invention features a method of screening for adiabetogene including: (a) supplying a cDNA library enriched fordiabetes-specific cDNA molecules; (b) supplying a cDNA library enrichedfor normal cDNA molecules; and (c) hybridizing the normal-enriched andthe diabetes-enriched cDNA libraries with a probe. Differentialhybridization of the probe to the normal-enriched and thediabetes-enriched libraries is indicative of a diabetogene. In preferredembodiments the method further includes hybridizing any differentiallyhybridizing probe from step (c) to RNA from a diabetic individual andRNA from a non-diabetic individual to confirm the differentialexpression of the differentially hybridizing probe.

Substantially pure DNA is DNA that is not immediately contiguous withboth of the coding sequences with which it is immediately contiguous(i.e., one at the 5' end and one at the 3' end) in thenaturally-occurring genome of the organism from which the DNA of theinvention is derived. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA or a genomic DNA fragment produced by PCR or restrictionendonuclease treatment) independent of other DNA sequences. It alsoincludes a recombinant DNA which is part of a hybrid gene encodingadditional polypeptide sequence.

Homologous refers to the sequence similarity between two polypeptidemolecules or between two nucleic acid molecules. When a position in bothof the two compared sequences is occupied by the same base or amino acidmonomeric subunit, e.g., if a position in each of two DNA molecules isoccupied by adenine, then the molecules are homologous at that position.The homology between two sequences is a function of the number ofmatching or homologous positions shared by the two sequences. Forexample, 6 of 10, of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology.

A transgene is defined as a piece of DNA which is inserted by artificeinto a cell and becomes a part of the genome of the animal whichdevelops from that cell. Such a transgene may be partly or entirelyheterologous to the transgenic animal.

A transgenic animal, e.g., a transgenic mouse, is an animal having cellsthat contain a transgene, which transgene was introduced into theanimal, or an ancestor of the animal, at an embryonic stage.

A substantially pure preparation of a polypeptide is a preparation whichis substantially free of the proteins with which it naturally occurs ina cell.

The invention is useful for: investigating the basis of glucosemetabolism in normal and disease states at the levels of gene structureand gene expression (mRNA or protein); determining if a subject is atrisk for a glucose-metabolism related disorder, e.g., diabetes, e.g.,Type II diabetes; evaluating the effect of a treatment, e.g., atreatment for a glucose-metabolism related disorder, e.g., diabetes, onthe level of expression of a diabetogene; evaluating animal models onthe basis of whether, at the level of diabetogene expression, the modelresembles a human disorder; and making transgenic animals andgenetically altered cell lines for use in research.

Other features and advantages of the invention will be apparent from thefollowing description and from the claims.

DETAILED DESCRIPTION

The drawings are first briefly described.

Drawings

FIG. 1 is a diagram of the screening method of the invention.

FIG. 2 is a diagram of theoretical patterns of diabetogene expression.

FIG. 3 is the cDNA and deduced amino acid sequence of human rad.

PREPARATION OF LIBRARIES

Preparation of Normal and type II cDNA Libraries

cDNA libraries were prepared using skeletal muscle mRNA from one normaland one Type II diabetic individual, see Sambrook et al. 1989 MolecularCloning: A Laboratory Manual Cold Spring Harbor Press, N.Y. The decisionto use single individuals for library construction was based on thepossible heterogeneity of Type II diabetes and fear thatgenetically-related alterations in gene expression in this disease couldbe obscured by pooling of samples. The cDNA was prepared using oligo-dTprimers and ligated into Lambda-Zap II (Stratagene) using EcoRIadapters. Lambda-Zap II contains the coding sequences for Bluescriptflanked by the filamentous phage initiation and termination sequences,allowing in vivo excision of the inserts in Bluescript in the presenceof a helper virus. The titers of the unamplified and amplified librarieswere ˜2×10⁶ and ˜2×10¹⁰, respectively, for both the diabetic and normalmuscle samples.

Muscle samples of a size sufficient for large scale RNA extraction wereobtained at surgery for amputation, frozen in liquid nitrogen andextracted for RNA using the guanidium isothiocyanate method.(Chomcyznski et al. 1987 Anal. Biochem. 162:156-159) Clinical data onthe metabolic state of the patient, the type and duration of diabetesand any complications which might exist were recorded. Great care wastaken to obtain the muscle samples as rapidly and cleanly as possible toavoid any degradation of mRNA. All samples were dissected from theviable margin of the limb from either quadriceps or gastrocnemiusmuscle. In humans, these muscles are comprised of both red and whitefibers, as well as other cell types (particularly vascular cells) whichmay have differing patterns of gene expression. (Klip 1990) Asindividual clones are selected in the subtraction process, it will beultimately important to validate that these are indeed products ofmuscle and that differences between diabetics and non-diabetics are notdue to differences in muscle type studied or to some underlyingcomplication of diabetes, such as uremia or infection. Since multiplesamples will be used for the screening Northern blots in which themuscle type and patient history are know, many of these differences (aswell as genetic differences in humans) should be detected.

Preparation of a library enriched in normal cDNAs and of a libraryenriched in Type II specific cDNAs

As initial step to identify genes whose expression was increased ordecreased in Type II diabetes, two subtraction libraries were prepared,one enriched in normal cDNAs and one enriched in diabetic cDNAs, using amodification of the procedure of Schweinfest, et al. (Schweinfest et al.1990 Genet. Anal. Techn. Appl. 7:64-70) and InVitrogen. The overallstrategy is outlined in FIG. 1. Initially, single-stranded DNA wasprepared from the normal and the Type II cDNA libraries using R408helper phage. The single-stranded DNA was isolated by polyethyleneglycol precipitation and purified by repeated phenol, chloroformphenoland chloroform extractions and, in some cases, further purified on aCsCl gradient. (Druguid et al. 1988 Proc. Natl. Acad. Sci. USA85:5738-5742) When analyzed by agarose gel electrophoresis and ethidiumbromide staining, the ss-DNA migrated a broad band from 1.6-4 kbreflecting the range of size of ss-pBluescript plus varying lengthinserts. In addition there was a discrete band at ˜4 kb representing thehelper phage DNA. A total of 120-150 μg of ss-DNA was isolated from eachcDNA library.

Approximately 60 μg of each ss-DNA preparation was biotinylated usingPhotoprobe biotin (Vector Laboratories). Specifically, 30 μg of DNA wasdissolved in 30 μl Hepes-EDTA buffer, sonicated for 1 minute and boiledfor 3 min to shear and denature the DNA, mixed with 60 μl Photoprobebiotin (0.5 mg/ml), and exposed to light from a 275W sunlamp at 10 cmfor 15 min. The reaction was quenched by addition of Tris-HCl, and theunreacted biotin removed by repeated extraction with 2-butanol. Thebiotinylated DNA was precipitated with ethanol and resuspended in dH₂ O.

To prepare a library enriched for genes preferentially expressed innormal muscle, 3 μg of ss-DNA from normal muscle was mixed with 30 μg ofbiotinylated ss-DNA from the diabetic muscle along with 3 μg of polyAand polyC and co-precipitated with ethanol on dry ice. The resultantpellet was resuspended in 10 μl dH₂ O, diluted with an equal volume of2× hybridization buffer (In Vitrogen) and incubated at 68° for 40-48hours. The hybrids of normal-diabetic DNA, as well as the unhybridizedbiotinylated "diabetic" DNA, were then removed using two extractionswith streptavidin and one extraction with Vectrix Avidin (avidin coupledto polysaccharide polymer). The enriched normal ss-DNA was thensubjected to a second round of subtraction using the same protocol.Based in studies using labeled DNA, two rounds of subtraction with a10-fold excess of DNA produce a ˜95% depletion of cDNA species common toboth libraries and a 20-fold enrichment of selected cDNAs.

A second subtraction library enriched for genes preferentially expressedin diabetic muscle was prepared in an analogous manner using ss-DNA fromdiabetic muscle and biotinylated ss-DNA from the normal muscle library.Both subtracted libraries were then converted to double strandedlibraries by incubation with DNA polymerase and ligase in the presenceof an appropriate primer (T3) and a mixture of oligonucleotides.

A library from a Type I diabetic, as well as a muscle library from anormal or diabetic individual using random primed cDNA can also be made.The Type I library could be used as the subtractor with the Type IIlibrary to help isolate cDNAs whose expression is altered in Type IIdiabetes specifically. Subtraction is most efficient using oligo-dTlibraries since all cDNAs have the same "relative starting point". Anormal muscle library prepared using random primers (rather thanoligo-dT) would be useful for getting full length sequence of clonesdetected in oligo-dT libraries. As described above the cDNA librariescan be constructed using Lambda Zap II with EcoRI adapters.

Availability of the Type I muscle library will allow preparation ofadditional subtraction libraries and/or subtraction probes as describedherein. Further modifications of the screening techniques describedherein can help narrow down the clones for screening and therefore yieldmore important clones. For example, the Type II diabetes-enrichedlibrary would be more likely to yield specific Type II "diabetogenes" ifthe subtraction is conducted using a Type I diabetic ss-DNA preparationagainst a Type II diabetic ss-DNA preparation, rather than a ss-DNApreparation from a non-diabetic individual. In addition, it may bedescribable to increase the number of rounds of substraction to furtherenhance the library for diabetes-related genes, and to use a Type Idiabetes enriched subtractive probe to further screen the dot-blots.(see below)

Screening for Diabetogenes

Initial Screening Strategy

An aliquot (1/40) of each subtraction library was used to transformcompetent XL-1 Blue cells (Stratagene) and plated on a media enrichedwith ampicillin, X-Gal and IPTG. For the "normal-enriched" library thisaliquot of the library yielded about 700 colonies; for the"diabetes-enriched" library this aliquot yielded about 450 colonies.Thus, the calculated library sizes are ˜28,000 and ˜18,000 for the"normal" and "diabetes" enriched libraries, respectively. In both casesabout 87% of the colonies were white indicating the presence of inserts.Duplicate colony lifts were prepared using BioTrans nylon membranes(ICN) form each dish of cells. One lift was hybridized to a cDNA probeprepared from the normal RNA and the other to a cDNA probe prepared fromthe diabetic RNA. (Sambrook 1989) About 30 colonies of interest weredefined in the aliquot of the normal-enriched library by differentialhybridization, and 19 colonies with differential hybridization wereidentified in the aliquot of the diabetes-enriched library. Each ofthese was then picked and expanded to a 5 ml mini-prep culture. DNA wasisolated using the method of Maniatas (Sambrook 1989), subjected to arestriction digest with EcoRI and analyzed by electrophoresis in 0.7%agarose. Almost all isolates contained inserts, varying in size from0.4-2 kb. The gels of these restriction digests were prepared induplicate and subjected to Southern blotting for an additional round ofdifferential screening using cDNA probes from the normal and diabeticRNA samples. Some inserts hybridized well to both probes, somehybridized poorly to both probes and some showed differentialhybridization suggestive that these were truly representative of genesdifferentially expressed in two muscle samples.

It was clear from preliminary experiments that the most difficult andtime consuming portion of this method is the screening of thesubtraction library for clones of interest. Two of the major limitationsof subtraction library analysis have already been alluded to and are (a)the failure of the procedure to remove completely some of the mostabundant cDNA species and (b) the difficulty in identifying very raremessages with this approach. Two (or even three) rounds of subtractionmay not eliminate common messages (which may still account for asignificant percentage of the subtraction clones), and it is quitepossible that some of the messages of interest may be non-abundant (suchas receptors or signaling molecules). To help overcome these problems,the screening strategy was modified as described below, by usingsubtracted probes to analyze replicate dot blots of the colonies of thelibrary. (Tindall et al. 1988 Biochemistry 27:6008-6013; Erlich 1989 PCRTechnology: Principles and Applications for DNA Amplifications. M.Stockton Press, N.Y.)

Improved Dot-Blot Screening Strategy

During the initial screening process, several technical problems wereidentified which appeared to limit the application of this method andproduce false positives, as well as false negatives. First, althoughduplicate colony lifts can be produced from a single plate, they tend tobe quite variable as to the amount of the colony (and hence cDNA) whichadheres to the filter. Using a velvet template to replicate plate thedishes improved reproducibility somewhat but was still suboptimal.Secondly, the bacterial plates were not optimal for long-term storage.After the potential positives were identified, and picked, the platescould be kept at 4° C. for several weeks, but tended to deteriorate orexhibit contamination if kept for months. Thus, except for the colonieswhich were picked, the library was lost for further screening. A thirdproblem was the inability of differential screening to identify rarermRNAs (cDNAs) in the library which might represent differentiallyexpressed and metabolically important species, since screening withtotal cDNA probes favors finding differences in more abundant mRNAspecies. A fourth problem, related to the two previous problems, wasthat since the clones not picked could not be stored for long periods oftime, it was impossible to return to the library to search for the cDNAsrepresenting rarer mRNAs which were indeed differentially expressed.

To help overcome these problems, a colony screening strategy wasdeveloped which provided for good duplicate filters, long-term storageof all colonies, and improved sensitivity for low abundance DNAs. Thus,after transformation, each colony was picked using a sterile toothpickand used to inoculate a single well of a 96-well plate. After initialgrowth, a replicate of this "archive" plate was made by the inoculationof bacterial cells from each well of the plate using a 96-wellreplicator beaded lid (FAST system, Falcon). The wells of the archiveplate were then diluted to a final concentration of 20% glycerol, andthe plates stored at -70° C. Duplicate dot-blots were prepared from thecopy plate (after allowing the infected bacterial to grow to highdensity) using a dot-blot manifold and BioTrans nylon membranes (ICN).

To overcome the difficulty in identifying rare messages, a method wasdeveloped to screen the dot-blots of the subtracted libraries usingsubtracted probes. Beginning with a small aliquot of dsDNA or ssDNA ofeach of the subtraction libraries as templates, subtracted probes wereprepared by the polymerase chain reaction (PCR) using SK and KS primers(since all inserts are in pBluescript). Under the proper conditions oftime and concentration, there was no difficulty in obtaining very highefficiency amplification of inserts up to 2 kb in size which can then becut with EcoRI to remove the polylinker and used as templates for randomprime labeling to give high specific activity subtracted probes. Tominimize the background created by some labeling of the polylinker,unlabeled pBluescript DNA digested with KpnI was added to thehybridization buffer, or the polylinker was removed from the EcoRIdigests using spin columns.

Duplicate dot-blots, from the "diabetic-enriched" colonies hybridizedwith a "normal" subtracted probe and a "diabetic" subtracted probe,(right side) were prepared. Very good quality replicate filters wereobtained. Clones which were strongly positive with the diabetic probeand negative with the normal probe were found. Screening of about 1/6 ofthe two subtraction libraries (this represents ˜2,000 colonies from the"normal" enriched library and ˜1,700 colonies from the diabetes enrichedlibrary) resulted in the discovery of 23 normal and 21 diabetic colonieswhich appeared to be differentially expressed. About half of these (11normal and 10 diabetic) were regarded as highly positive and had insertson the plasmid preparation. In addition, on this initial screen about190 and 140 colonies from each library gave a signal too low on bothfilters to determine possible differential expression. These presumablyrepresent less abundant mRNAs which may or may not be differentiallyexpressed but may represent some of the most interesting cDNAs from ametabolic point of view. Since all of the colonies have been saved inthe archive plate, it will now be possible to prepare fresh duplicatedot-blot filters to analyze these less abundant mRNAs/cDNAs.

Analysis of Differential Clones for Insert and Differential Expressionby Northern Blots

Following identification on the dot blots using the subtracted probes,each of the candidate clones was expanded to a 30 ml culture, and theplasmid DNA isolated and analyzed by restriction digest with EcoRI. Thisyields sufficient DNA for electrophoretic analysis, purified insert forlabeling of up to 4 Northern blots and sequence analysis. The insertsvaried in size from 0.4 to 2.0 kb (Table 1) and were purified by agarosegel electrophoresis and Gene Clean II. The most important step ofinitial characterization of these subtraction libraries is to determinewhich of these presumptive differential clones are truly representativeof "diabetes-related" changes in gene expression, which are simply dueto individual variation and which are specific to Type II versus Type Idiabetes. To this end, screening Northern blots were prepared using RNA(20 μg) samples from several normal and diabetic individuals (both TypeII and Type I). These blots can be hybridized using random primed probesprepared from the isolated inserts of the differential colonies todetermine the true pattern of expression.

A schematic showing the various predicted patterns of expression isshown in FIG. 2. Five types of patterns were expected and areillustrated in this schematic. Some probes will fail to showdifferential hybridization at this stage despite showing differentialproperties in the earlier stages of screening (Pattern E). Theserepresent mRNAs which are variable in the population or abundant mRNAswhich were not removed by subtraction, but are not truly differentiallyexpressed. Other probes might recognize messages preferentiallyexpressed in normals (Pattern A), mRNAs preferentially expressed in bothTypes I and II diabetics (Pattern B), or RNAs preferentially expressedin some or all Type II diabetics only (Patterns C and D). These patternshelp to identify those mRNA species whose expression might be altered inall patients with diabetes, and this represent some general metabolicalteration, versus those differences in gene expression which areprimarily due to insulin deficiency and hence expressed predominantly inType I diabetes or due to insulin resistance and expressed predominantlyin Type II diabetes. Some clones would be expected to show variableexpression in the diabetics if the expression is related to level ofcontrol, or other metabolic or genetic variables.

Of the initial 21 clones studied at the Northern stage, 2 were enrichedin normals (for example clone G5N7) and 7 were enriched in diabetes (forexample, clones B10D6 and C9D6). (See Table I) Note that clone B10D6appears to represent an mRNA species which is preferentiallyoverexpressed in all diabetics, where clone rad represents a speciesoverexpressed primarily in Type II diabetics.

Despite all previous efforts at selection, approximately 30% of cloneswere non-differential at this stage.

                  TABLE I                                                         ______________________________________                                        Potential Diabetes-Related Clones                                                     Insert   mRNA                                                         Identi- Size.sup.‡                                                                  size     Expression                                                                            Sequence/                                   fication                                                                              (kb)     (kb).sup.†                                                                      in Diabetes                                                                           Homology                                    ______________________________________                                        B1D4    0.7      2.6 (4.6)                                                                              Increased                                                                             Muscle glycogen                                                               phosphorylase                               B10D6   1.5      2 (4.2)  Increased                                                                             Human elongation                                                              factor 1α                             G2D8    0.5      3.3, 2.5 Increased                                                                             No hit in                                                                     GeneBank                                    B14D7   1.7      N.D.     Increased                                                                             not done                                    F2D8    0.4      2.8 (6.4)                                                                              Increased                                                                             mitochondral                                C9D6 ( rad)                                                                           0.8      3.8, 1.4 Increased                                                                             no hit in                                                                     Genebank                                    C8N8    0.4      2.8 (6.4)                                                                              Increased                                                                             Same as F2D8                                N13     0.4      7, 1.6   Decreased                                                                             Human myosin                                G5N7    1.0      ˜1 kb                                                                            Decreased                                                                             Human myoglobin                             D55     1.0      7.0      Variable                                                                              No hit in Gene                                                                Bank                                        C12N2   1.0               Variable                                                                              No hit in Gene                                                                Bank                                        D5N4*   1.8               Variable                                                                               E. coli β-                                                              galactosidase                               C1N4    1.0               Variable                                                                              E. coli β-                                                               galactosidase                               D5N3    0.2, 0.4,         Variable                                                                              Alu a                                               0.7                                                                   A8N6    1.1               Variable                                                                              Not done                                    C4N7    0.5               Variable                                                                              Not done                                    C5D7    2.0               Variable                                                                              Not done                                    ______________________________________                                         *six other similar clones identified                                          .sup.† minor species are in parenthesis                                .sup.‡ EcoRI digests                                          

Characterization of Diabetes-Related Clones

Once a clone which shows one of the "diabetes-related" patterns byNorthern analysis described above has been identified characterizationwill include sequencing, comparison to genetic data banks for homologyor identity to know DNA and protein sequences, analysis of pattern ofexpression in human and rodent tissues, and ultimately preparation ofanti-peptide antibodies for study of protein expression and function.Depending on the number of such clones, it may be desirable toprioritize their evaluation. Prioritization will largely depend oninformation obtained in the screening Northern blots. Highest prioritycan be given to a given class e.g., to the class of clones expresseddifferentially in Type II diabetes only (such as clone C9D6 in Table I)on clones showing the greatest differential levels of expression.

Clones can be sequenced on both strands with Sequenase (United StatesBiochemicals) using initially T3 and T7 primers. Other specific primersselected at convenient intervals to allow construction of a completecDNA sequence can be used. It is likely that most inserts will notrepresent full length clones, and thus, to obtain full-length sequenceof interesting clones, it will be necessary to screen the originallibraries and the random-primed muscle cDNA library for related cDNAs,and in turn, to sequence these. All sequences should be analyzed usingthe EUGENE and SAM programs.

Sequencing and Identification of Differential Clones

Potential positive clones from the screens described above were expandedto 30 ml cultures and the plasmid DNA were isolated using Qiagenmini-prep columns. The DNA was then digested by EcoRI and the insertsize determined by agarose gel electrophoresis as described above.Partial sequence data was then obtained from the isolated DNA using T3and T7 primers and Sequenase (United States Biochemicals).

A summary of the insert size, differential expression, and sequence datais presented in Table I. Thus far, limited sequence data are availableon 9 clones, all of which have been compared to genetic data banks forhomology to known DAN and protein sequences using the SAM and EUGENEprograms.

Two of the clones over-expressed represent metabolic enzymes notpreviously studies at the RNA level in diabetes. One (B1D4) is 96%homologous with rat muscle glycogen phosphorylase; other (B10D6) is >95%identical with human elongation factor 1α. Two clones which showmoderate decreases in expression in diabetes have 100% homology withabundant muscle proteins, myosin and myoglobin. Finally, three of thedifferently expressed cloned show no match in the GeneBank. Two of thefrequently observed "false positives" were also sequenced forinformation. One was identified as a Bluescript clone in which a partialduplicated β-galactosidase gene (E. coli.) was acting as an insert dueto some gene rearrangement and the other as an Alu sequence.

Diabetogenes in Other Species

Diabetogenes can be isolated from other species by methods analogous tothose described above. Alternatively, once a diabetogene has beenisolated in a first species, e.g., in humans, it can be isolated from asecond species by methods known to those skilled in the art. Forexample, murine Diabetogene rad can be isolated by probing a mouse cDNAor genomic library with a probe homologous to human Diabetogene rad.

Human Diabetogene rad

Clone C9D6 (isolated as described above) corresponds to a previouslyunknown human gene, rad. The expression of Diabetogene rad is increasedin patients with NIDDM but not in patients with IDDM. Northern blotanalysis of three normals, two Type I diabetics and five Type IIdiabetics was performed. Four of the five type II diabetics exhibitsignificant increases in mRNA for this interesting gene as compared toboth the Type I diabetic and non-diabetic individuals. This result wasreproducible in other blots.

The DNA sequence (and deduced amino acid sequence) of rad is shown inFIG. 3. (Sequence ID No. 1). The sequence of the rad gene includes aninitiator codon at Met¹²² and Met²⁴¹. Proteins (and DNA sequencesencoding them) beginning at either site are within the invention,although it is believed that the in vivo initiation site is at Met²⁴¹.This sequence does not match any known sequence in GenBank, although onedomain has some features suggestive of homology to a known transcriptionfactor.

The cloned sequences can be expressed in an expression system to yieldrecombinant rad polypeptide. Antibodies, preferably more clonalantibodies directed against the Diabetogene rad protein can be made bymethods known to those skilled in the art.

Other Human Diabetogenes

Several diabetogenes isolated by the methods described above correspondto known genes with known functions.

The expression of three of these genes or sequences, muscle glycogenphosphorylase (corresponding to clone B1D4), human elongation factor is(corresponding to clone B10D6), and a segment of the human mitochondrialgenome which codes for cytochrome oxidase and several transfer RNAs(corresponding to clone F2D8), has been shown to be elevated in Type Iand Type II diabetes.

Clone B1D4 has been shown to represent human muscle glycogenphosphorylase. The mRNA for this important enzyme of glycogen metabolismis increased in skeletal muscle of both Type I and Type II diabeticpatients and also increased in muscle of streptozotocin diabetic rats.Studies of the regulation of this gene in muscle cell lines (L6) intissue culture by both insulin and glucose and will correlate thesechanges with protein and enzyme activity. These changes in glycogenphosphorylase may contribute to the changes in glycogen synthesisactivity which are observed in diabetes.

Clone B10D6 has been identified as human elongation factor is (EF1α).This is a GTP binding protein which plays a critical role in the earlysteps of protein synthesis and ribosome formation, but whose regulationin disease has not yet been studied. In additional Northern blotanalysis has been shown that the level of message is increased in muscleof individuals with both Type I and Type II diabetes between 5- and10-fold when compared to normal. In a muscle sample from one Type Idiabetic who had previously undergone pancreatic transplantation, thelevel of EF1α was decreased toward normal. EF1α message has also beenshown to be increased in muscle and liver of streptozotocin diabeticrats but not muscle of ob/ob mice.

Clone F2D8 was previously identified to be increased in both Type I andType II diabetes. Preliminary sequence analysis of this clone shows itto be 97% identical to a segment of the human mitochondrial genome whichcodes for cytochrome oxidase and several transfer RNAs. In view of thevery recent description of a patient with a form of diabetes related toa defect in mitochondrial gene transmission this finding is of greatinterest.

Use

The peptides of the invention may be administered to a mammal,particularly a human, in one of the traditional modes (e.g., orally,parenterally, transdermally, or transmucosally), in a sustained releaseformulation using a biodegradable biocompatible polymer, or by on-sitedelivery using micelies, gels and liposomes or by transgenic modes.

Other Embodiments

The level of expression of one, or a combination, of diabetogenes can beused in the investigation of normal, experimentally perturbed, ordisease-state metabolism. For example, the effect of a treatment, e.g.,the administration of a drug, can be studied by its effect ondiabetogene expression. This approach could be used to optimize ormonitor treatment, or to assist in the discovery or evaluation of newtreatments, e.g., of new drugs.

The effect of a treatment, e.g., the administration of a drug, could beevaluated by its effect on diabetogene mRNA or protein levels in: ananimal, e.g., a human, or in an experimental animal, e.g., a transgenicanimal, e.g., a transgenic animal with a mutant Diabetogene radtransgene, or an animal which is a "diabetes model", e.g , a NOD mouse,an ob/ob mouse, a db/db mouse, a Zucker fatty rat, or a streptozotocininduced rat; or a cultured cell or tissue. A useful therapy would be onewhich, e.g., would shift diabetogene expression in the direction of wildtype.

Diabetogene expression patterns could also be used to evaluate new orexisting animal-diabetes models, where a desirable model would have apredetermined pattern of diabetogene expression, e.g., a pattern whichresembles that seen in a human disease state. Similarly, the effect of amutation in a gene, e.g., a gene involved in glucose metabolism, couldbe studied by comparing diabetogene expression in wild type and mutantcells e.g., cell lines, or in wild type and mutant organisms.

Nucleic acid encoding a diabetogene can be used to transform cells. Forexample, the Diabetogene rad, e.g., a mutant form of the gene, e.g., adeletion, can be used to transform a cell and to produce a cell in whicha copy of the cell's genomic Diabetogene rad has been replaced by thetransformed gene, producing, e.g., a cell deleted for a copy of thegene. This approach can be used with cells capable of being grown inculture, e.g., cultured muscle cells, to investigate the function of thegene.

A diabetogene, e.g., Diabetogene rad, e.g., a mutant form of the gene,e.g., a deletion, can be used to transform a cell which subsequentlygives rise to a transgenic animal. This approach can be used to create,e.g., a transgenic animal in which a copy of the gene is inactivated,e.g., by a deletion. Homozygous transgenic animals can be made bycrosses between the offspring of a founder transgenic animal.Genetically altered cell lines can be made from transgenic animals. Suchanimals and cell lines would be useful in research.

The pattern of expression of one or a combination of diabetogenes couldbe used to diagnose persons at risk for diabetes or other disorders.This could be accomplished by monitoring expression of diabetogene mRNAor protein. Diabetogene rad, e.g., is more highly expressed in Type IIdiabetics than in Type I diabetics or in normal individuals, thus itsexpression could be used as a means of diagnosing risk for Type IIdiabetics.

Individuals at risk for a glucose-related disorder can be identified bythe possession of structural defects in a diabetogene. For example,nucleic acid from an individual can be analyzed for gross chromosomalrearrangements, e.g., deletions, insertions, or translocations, or frameshifts, or point mutations at a diabetogene, by blot or sequenceanalysis. Even if critical diabetogenic lesions have not beencharacterized any gross rearrangement or any lesion which changed thereading frame of the gene would be highly likely to alter function. Inthe case of Diabetogene rad, any lesion likely to increase expressionwould be highly likely to induce a disease state.

The invention also includes preparations which inhibit the function of adiabetogene and/or its products, e.g., antibodies or antisense nucleicacids.

The invention includes the protein encoded by Diabetogene rad or anyprotein which is substantially homologous to Diabetogene rad or to thefragment of Diabetogene rad described in FIG. 3 (Seq ID No. 1) as wellas other naturally occurring diabetogenes. Also included are: allelicvariations; natural mutants; induced mutants, e.g., in vitro induceddeletions; proteins encoded by DNA that hybridizes under high or low(e.g., washing at 2×SSC at 40 C. with a probe length of at least 40nucleotides) stringency conditions to a nucleic acid naturally occurring(for other definitions of high and low stringency see Current Protocolsin Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1-6.3.6,hereby incorporated by reference); and polypeptides or proteinsspecifically bound by antisera to the Diabetogene rad protein,especially by antisera to the active site or binding domain of theDiabetogene rad protein. The term also includes chimeric polypeptidesthat include the Diabetogene rad protein.

The invention also includes any biologically active fragment or analogof the Diabetogene rad protein. By "biologically active" is meantpossessing any in vivo or in vitro activity which is characteristic ofthe Diabetogene protein.

Preferred analogs include Diabetogene rad analogs (or biologicallyactive fragments thereof) whose sequences differ from the wild-typesequence only by conservative amino acid substitutions, for example,substitution of one amino acid for another of the same class (e.g.,valine for glycine, arginine for lysine, etc.) or by one or morenon-conservative amino acid substitutions, deletions, or insertionswhich do not destroy the polypeptide's biological activity. Preferredanalogs also include Diabetogene rad proteins (or biologically activefragments thereof) which are modified for the purpose of increasingpeptide stability; such analogs may contain, for example, one or moredesaturated peptide bonds or D-amino acids in the peptide sequence.

Analogs can differ from naturally occurring Diabetogene rad protein byamino acid sequence differences or by modifications that do not affectsequence, or by both. Analogs of the invention will generally exhibit atleast 70%, more preferably 80%, more preferably 90%, and most preferably95% or even 99%, homology with all or part of a naturally occurringDiabetogene rad protein sequence. The length of comparison sequenceswill generally be at least about 20 amino acid residues, preferably morethan 40 amino acid residues. Modifications include, or in vitro chemicalderivatization of polypeptides, e.g., acetylation, or carboxylation.Also included are modifications of glycosylation, e.g., those made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing or in further processing steps, e.g., byexposing the polypeptide to glycosylation affecting enzymes derived fromcells that normally provide such processing, e.g., mammalianglycosylation enzymes. Also embraced are versions of the same primaryamino acid sequence that have phosphorylated amino acid residues, e.g.,phosphotyrosine, phosphoserine, or phosphothreonine. Analogs can differfrom naturally occurring Diabetogene rad protein by alterations of theirprimary sequence. These include genetic variants, both natural andinduced. Also included are analogs that include residues other thannaturally occurring L-amino acids, e.g., D-amino acids or non-naturallyoccurring or synthetic amino acids, e.g., β or γ amino acids.Alternatively, increased stability may be conferred by cyclizing thepeptide molecule.

In addition to substantially full-length polypeptides, the inventionalso includes biologically active fragments of the polypeptides. As usedherein, the term "fragment", as applied to a polypeptide, willordinarily be at least about 10 contiguous amino acids, typically atleast about 20 contiguous amino acids, more typically at least about 30contiguous amino acids, usually at least about 40 contiguous aminoacids, preferably at least about 50 contiguous amino acids, and mostpreferably at least about 60 to 80 or more contiguous amino acids inlength. Fragments of Diabetogene rad protein can be generated by methodsknown to those skilled in the art. The ability of a candidate fragmentto exhibit a biological activity of Diabetogene rad protein can beassessed by methods known to those skilled in the art. Also included areDiabetogene rad protein polypeptides containing amino acids that arenormally removed during protein processing, including additional aminoacids that are not required for the biological activity of thepolypeptide, or including additional amino acids that result fromalternative mRNA splicing or alternative protein processing events.

The invention also includes nucleic acids which encode the polypeptidesof the invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1443                                                              (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GTGGCTGCAGCAGCAGCGGCGGCGGAAACCCTAAAGTCCGAGTCCGGACTACGAGTGCGT60                GGCCTCCTAATCCGGATCCTAGTCCTGAGCGTGTCTGTGTGCGAGTGGACGGTCCCGGAC120               GCG123                                                                        ATGACCCTGAACGGCGGCGGCAGCGGAGCGGGCGGGAGCCGCGGTGGG171                           metthrleuasnglyglyglyserglyalaglyglyserargglygly                              151015                                                                        GGCCAGGAGCGCGAGCGCCGTCGGGGCAGCACACCCTGGGGCCCCGCC219                           glyglngluarggluargargargglyserthrprotrpglyproala                              202530                                                                        CCGCCGCTGCACCGCCGCAGCATGCCGGTGGACGAGCGCGACCTGCAG267                           proproleuhisargargsermetprovalaspgluargaspleugln                              354045                                                                        GCGGCGCTGACCCCGGGTGCCCTGACGGCGGCCGCGGCCGGGACGGGG315                           alaalaleuthrproglyalaleuthralaalaalaalaglythrgly                              505560                                                                        ACCCAGGGTCCCAGGCTGGACTGGCCCGAGGACTCCGAGGACTCGCTC363                           thrglnglyproargleuasptrpprogluaspsergluaspserleu                              65707580                                                                      AGCTCAGGGGGCAGCGACTCAGACGAGAGCGTTTACAAGGTGCTGCTG411                           serserglyglyseraspseraspgluservaltyrlysvalleuleu                              859095                                                                        CTGGGGGCGCCCGGCGTGGGCAAGAGCGCCCTGGCGCGCATCTTCGGC459                           leuglyalaproglyvalglylysseralaleualaargilephegly                              100105110                                                                     GGTGTGGAGGACGGGCCTGAAGCAGAGGCAGCAGGGCACACCTATGAT507                           glyvalgluaspglyproglualaglualaalaglyhisthrtyrasp                              115120125                                                                     CGCTCCATTGTAGTGGACGGAGAAGAGGCATCACTCATGGTCTACGAC555                           argserilevalvalaspglygluglualaserleumetvaltyrasp                              130135140                                                                     ATTTGGGAGCAGGACGGGGGCCGCTGGTTGCCCGGCCACTGCATGGCC603                           iletrpgluglnaspglyglyargtrpleuproglyhiscysmetala                              145150155160                                                                  ATGGGGGATGCCTATGTCATTGTGTACTCAGTGACGGACAAGGGCAGC651                           metglyaspalatyrvalilevaltyrservalthrasplysglyser                              165170175                                                                     TTCGAGAAGGCCTCAGAACTGCGGGTCCAGCTGCGGCGTGCACGGCAA699                           pheglulysalasergluleuargvalglnleuargargalaarggln                              180185190                                                                     ACAGATGATGTGCCCATCATCCTCGTGGGCAACAAGAGCGACCTGGTG747                           thraspaspvalproileileleuvalglyasnlysseraspleuval                              195200205                                                                     CGCTCTCGTGAGGTCTCGGTGGATGAGGGCCGGGCCTGCGCGGTGGTC795                           argserarggluvalservalaspgluglyargalacysalavalval                              210215220                                                                     TTTGACTGCAAGTTCATTGAGACATCAGCGGCATTGCACCACAATGTC843                           pheaspcyslyspheilegluthrseralaalaleuhishisasnval                              225230235240                                                                  CAGGCGCTGTTTGAAGGTGTCGTGCGCCAGATACGCCTGCGCAGGGAC891                           glnalaleuphegluglyvalvalargglnileargleuargargasp                              245250255                                                                     AGCAAAGAAGCCAACGCACGACGGCAAGCAGGCACCCGGAGGCGAGAG939                           serlysglualaasnalaargargglnalaglythrargargargglu                              260265270                                                                     AGCCTTGGCAAAAAGGCGAAGCGCTTCTTGGGCCGCATCGTAGCTCGT987                           serleuglylyslysalalysargpheleuglyargilevalalaarg                              275280285                                                                     AACAGCCGCAAGATGGCCTTTCGCGCCAAATCCAAGTCCTGCCACGAC1035                          asnserarglysmetalapheargalalysserlyssercyshisasp                              290295300                                                                     CTCTCGGTTCTCTAG1050                                                           leuservalleu                                                                  305                                                                           GTCCCACCCGCTCCCACTATGGTGGGAGACGAACGGAAGGGTTGGTGGGCTGGCCCAGCC1110              AACTGCCCCGGGTGCCTCAGAGCAGGCTCAGACTCTGGGTCCCTCGGAGCTGCCAGCCGG1170              GCACCCCCAACCTCATGGTCATGGACAGATAGACAGTGCTGCCCTGCGAAGTGGCTCTCA1230              GGGGCCAGTGAGGGCTGGGCCCACAGAGATGCATGCGCAGGCTCATATGCGTCCCAAGCA1290              GCCGCAGCGCAGCCGCCGGGCAGGCCTGCGTGCCGGGAGAGGACTCTGCCTTTTTTCACA1350              GCCCGGGTGTGCCTGCCCTGGAGGGAGGCTCTTCAGTGCGGTAGCTACTTGTTTACATGC1410              AGATTTTTGTAATAAAGGCTATTTCCTGATAAA1443                                         __________________________________________________________________________

Other embodiments are within the following claims.

What is claimed is:
 1. Purified DNA comprising a sequence encoding humanrad.
 2. A vector comprising a DNA sequence encoding human rad.
 3. Acultured cell containing the purified DNA of claim
 1. 4. The culturedcell of claim 3, wherein said cell is a muscle cell.
 5. The culturedcell of claim 3, wherein said cell expresses human rad.
 6. A homogeneouspopulation of cultured cells, each of which comprises the purified DNAof claim
 1. 7. A method for manufacture of the human rad proteincomprising culturing the cultured cell of claim 3 in a medium to expresssaid protein.
 8. Purified DNA of claim 1 comprising a sequence encodingthe human rad polypeptide of SEQ ID NO:1.